The examination of samples by means of microscopy is a broad technical field for which there are varied technical solutions. Starting from the standard light microscopy, widely different microscopy methods have evolved.
A standard field of use of light microscopy for examining biological preparations is luminescence microscopy. In this process, particular dyes (so-called phosphors or fluorophores) are used for the specific tagging of samples, e.g. of cell parts. The sample is, as mentioned, illuminated with excitation radiation and the luminescence light excited thereby is recorded by suitable detectors. For this, a dichroic beam splitter is usually provided in the light microscope in combination with block filters which split the fluorescence radiation from the excitation radiation and enable a separate observation. Through this procedure, the imaging of individual, differently coloured cell parts in the light microscope is possible. Of course, several parts of a preparation can also be simultaneously coloured with different dyes attaching specifically to different structures of the preparation. This method is called multiple luminescence. Samples which luminesce per se, thus without added tagging substance, can also be surveyed.
Here, luminescence is understood, as is generally usual, as a generic term for phosphorescence and fluorescence, thus covers both processes.
To examine samples, it is also known to use laser scanning microscopy (also LSM for short) which, from a three-dimensionally illuminated image, reproduces by means of a confocal detection arrangement (when it is called a confocal LSM) or a non-linear sample interaction ( so-called multiphoton microscopy) only that plane which is located in the focal plane of the objective. An optical section is produced and the recording of several optical sections at different depths of the sample then allows the generation, with the help of a suitable data-processing device, of a three-dimensional image of the sample which is composed of the different optical sections. Laser scanning microscopy is thus suitable for examining thick preparations.
Of course, a combination of luminescence microscopy and laser scanning microscopy is also used, in which a luminescent sample is imaged at different depth levels with the help of an LSM.
Laser scanning microscopes are also supplied as retrofit modules for normal light microscopes which then also enable a laser scanning microscopy of samples in addition to a normal microscopy. Like standard microscopy, laser scanning microscopy, and in particular laser scanning microscopy of luminescent samples, is also characterized by the optical resolution limit of the microscope used. However, laser scanning microscopy allows a very much higher depth of field, although with both microscopes the optical resolution is diffraction-limited by physical laws.
Different approaches have recently been developed for resolutions beyond the diffraction limit. These microscopy methods are characterized by the fact that they provide the user with a higher lateral and/or axial optical resolution compared with the standard microscope. In this description, such microscopy methods are called high-resolution microscopy methods, as they achieve a resolution beyond the optical diffraction limit. Diffraction-limited microscopes, on the other hand, are called standard microscopes. They realize known optical wide-field microscopy or laser scanning microscopy.
The gain in resolution with high-resolution microscopy is at the cost of a lower measuring speed, a smaller measuring field, as well as limitations in respect of the penetration depth and the tagging substances that can be used for the luminescence microscopy.
Thus, high-resolution microscopy methods can be used only for particular examinations, because of the essential compromises in respect of sample preparation and acquisition parameters (in particular acquisition speed, etc.). This is particularly awkward if it is wished to examine smaller areas in a large sample or to track a temporal development of a sample which proceeds at varying speeds.